Study On The Role And Mechanism Of MiR-551b-5p In Severe Acute Pancreatitis Complicated With Capillary Leakage

Background and objective:Acute pancreatitis(AP)is one of the most common critical diseases of the digestive system,and its incidence is increasing.About one-fifth of patients with AP could develop severe acute pancreatitis(SAP)with high morbidity,which is one of the major diseases that endanger people’s health and life.The acute phase of SAP is often accompanied by Capillary leakage syndrome(CLS),which is the intermediate stage from systemic inflammatory response syndrome to multiple organ failure of SAP.Therefore,the treatment of SAP complicated with CLS has important clinical significance to improve the prognosis of SAP patients.Unfortunately,the mechanism of SAP complicated with CLS is still unclear,and there is a lack of effective treatment.At present,studies suggest that the mechanism of CLS is the damage of capillary endothelial cells,which leads to the abnormality of tight junctions,adherens junctions,and transcellular pathways between endothelial cells,increasing capillary permeability.Recent researches show that microRNAs(miRNAs)are closely related to the occurrence,development,and prognosis of pancreatic diseases.In our earlier study,miR-551b-5p was found to be associated with SAP complications.Therefore,this study aims to explore the mechanism of miR-551b-5p in SAP complicated with CLS by combining sequencing and luciferase technology at the cellular level and biological individual level.It will provide a new idea for understanding the mechanism of CLS in SAP.Materials and Method:1.To explore the relationship between miR-551b-5p and capillary leakage in SAP1.C57/BL6 mice were grouped randomly:control group,Cer group,and Cer+LPS group,with 8 mice in each group.The mice in the Cer group were given an intraperitoneal injection of Caerulein(Cer),and the mice in the Cer+LPS group were given an intraperitoneal injection of Cer+Lipopolysaccharide(LPS).2.24h after modeling,samples were obtained by dissecting each mouse per group,including serum,pancreas,lung,liver,kidney,colon,and fat.3.Biochemical analyzer was used to detect the differences in the expression of serum amylase and lipase biochemical indexes of the three groups.The expressions of IL-1β,TNF-α,and IL-6 in the serum of the three groups were detected by ELISA.4.Hematoxylin-eosin(HE)Staining was performed on the pancreas of each group of mice,and tissue damage was observed under the microscope.5.The changes in capillary endothelial cells of pancreatic were analyzed by transmission electron microscopy(TEM).6.The wet-dry weight ratio of the pancreas and lung of mice,and Evan’s blue concentration in tissues were detected to understand the differences in the degree of capillary leakage of the three groups.7.qPCR was applied to detect the expression of miR-551b-5p in serum,pancreas,lung,liver,kidney,colon,and adipose tissue of the three groups.2.To explore the role of miR-551b-5p in capillary leakage of SAP1.The acute inflammatory cell model was constructed.Then,the expression levels of inflammatory cytokines(TNF-α and IL-6)were detected by qPCR to determine the modeling efficiency.2.The expression of miR-551b-5p between the control group and the acute inflammatory cell model group was detected by qPCR.Meanwhile,mRNA and protein expression of AQP5,Occludin,JAM3,and Claudin1 were determined by qPCR and Western Blot(WB).3.miR-551b-5p mimic and miR-551b-5p inhibitor were constructed.The transient transfection was used to up-regulate and down-regulate the expression of miR-551b-5p in cells,and the efficiency of miR-551b-5p transfection in cells was detected by qPCR.4.Human umbilical vein endothelial cells(HUVECs)were grouped randomly:blank group,NC-mimic group,and miR-551b-5p mimic group.The permeability of cells was detected by fluorescence yellow.5.Phalloidin/FITC staining was used to detect the morphological differences of vascular endothelial cytoskeleton in each group.6.qPCR and WB were used to detect mRNA and protein expression differences of tight junction-related molecules(Occludin,JAM3,and Claudin1)and transcellular pathway molecule(AQP5)of HUVEC cells in the NC-mimic group and miR-551b5p mimic group.In addition,immunofluorescence was used to detect the expression of Occludin and JAM3.7.AAV8-miR-551b-5p and AAV8-miR-551b-5p Spone were constructed to up-regulate and down-regulate the expression of miR-551b-5p in mouse pancreas.Then,the pre-experiment was performed:AAV8 was injected into mice by intraperitoneal injection(IP)and caudal vein(Ⅳ)to screen out the best injection route for virus infection.And,the transfection efficiency of AAV8 was detected by qPCR and WB.8.Subsequently,formal animal experiments were performed:AAV8-NC and AAV8-miR-551b-5p were intraperitoneally injected into mice,and the SAP mouse model was constructed 3 weeks later to further explore the effects of miR-551b-5p on SAP with capillary leakage.24 hours after modeling,samples were collected and photographed for each group of mice:serum,pancreatic tissue and lung tissue.9.The expressions of IL-1β,TNF-α,IL-6,amylase,and lipase in the serum of each group were detected by ELISA and biochemical analyzer.10.The wet-dry weight ratio of the pancreas and lung,and Evan’s Blue concentration in tissues were determined to understand the degree of capillary leakage of mice in each group.11.The pancreas and lung histology and morphology were observed with HE staining Immunohistochemical staining was performed on the pancreatic tissues to observe the difference in MPO expression in the pancreatic tissues of each group.12.The mRNA and protein expression differences of tight junction-related molecules(Occludin,JAM3,and Claudin1)and transcellular pathway molecules(AQP5)of vascular endothelial cells in pancreatic tissue of mice in each group were detected by qPCR and WB.13.Immunofluorescence staining was used to detect the expression of Claudin1 and Occludin in the pancreas of mice in each group.3.To explore the mechanism of miR-551b-5p in SAP complicated with capillary leakage1.Preparation of sequencing cell samples:the transient transfection was used to up-regulate the expression of miR-551b-5p in HUVECs.Then,transfer efficiency was detected by qPCR.2.RNA-seq was performed on HUVECs of the mimic-NC group and miR-551b-5p mimic group to screen out differentially expressed genes.3.GO functional enrichment and KEGG signaling pathway enrichment analysis were performed on the differentially expressed genes.4.The sequenced differential genes were combined with Targetscan and miRBD online website tools to jointly predict the possible target genes of miR-551b-5p,and screen out the related signaling pathways of miR-551b-5p in SAP with CLS.5.The expression of predicted target genes in HUVECs and mice pancreas of the control and overexpressed miR-551b-5p groups were detected by qPCR,and the target gene of miR-551b-5p was verified by dual-luciferase reporter assay.6.WB was used to detect the protein expression differences of ERBB3 and the key molecules in the PI3K/AKT signaling pathway in cells and mouse pancreatic tissues of the control group,the overexpressed miR-551b-5p group,and the overexpressed miR-551b-5p reconstructed module.To explore the effect of miR-551b-5p on PI3K/AKT signaling pathway by targeting ERBB3.7.After purchasing 740Y-P(PI3K agonist),HUVECs were grouped randomly:mimic-NC group,miR-551b-5p mimic group,Mime-NC+740 Y-P group,and miR-551b-5p mimic+740 Y-P group.The protein expression differences of tight junction-related molecules(Occludin,JAM3,and Claudin1)and transcellular pathway molecules(AQP5)of HUVECs in the above groups were detected by WB.Results:1.miR-551b-5p was positively correlated with capillary leakage and disease severity in SAP mice1.Compared with the control group,the mice in the Cer and Cer+LPS groups had obvious pancreatic tissue lesions,and the serum amylase,lipase,TNF-α,IL-6,and IL-1β levels were significantly increased(P<0.05).After HE staining,it was observed under the microscope that there was no obvious pathological damage to the pancreatic tissue of mice in the control group,while there were obvious pathological changes in the pancreatic tissue of mice in the Cer and Cer+LPS groups,and the pathological score of the degree of damage was significantly increased(P<0.05).This suggests that the SAP models of mice in the Cer and Cer+LPS groups are stable.2.Compared with the Cer group,the Cer+LPS group had higher serum amylase,lipase,TNF-α,IL-6,IL-1β,and the pathological damage score of pancreatic tissue(P<0.05).In addition,the wet/dry ratio of pancreas and lung tissues and Evans blue leakage in the Cer+LPS group were higher than those in the Cer group(P<0.05).These results suggested that the severity of acute pancreatitis and capillary leakage in the Cer+LPS group were more severe than those in the Cer group.3、The results of TEM showed that compared with the control group,the Cer group and Cer+LPS group had changes in the capillary endothelial cells of pancreatic tissue.Compared with the Cer group,the damage of capillary endothelial cells and intercellular junction in the Cer+LPS group was more obvious.4、qPCR results showed that miR-551b-5p in the pancreas,lung,and serum of mice in the Cer+LPS group compared to the control group increased the most(P<0.05),followed by the Cer group(P<0.05).This suggests that the more severe the AP condition,capillary leakage,and capillary endothelial cell destruction in mice,the higher the expression of miR-551b-5p in pancreatic tissue,lung tissue,and serum.5、To explore the source of miR-551b-5p,qPCR results showed that in normal mice,the expression of miR-551b-5p in various organs from high to low was the serum,liver,pancreas,fat,kidney,lung,and colon.There was no significant difference in the expression of miR-551b-5p in the kidney among the three groups(P>0.05),but miR-551b-5p was significantly decreased in the adipose tissue of the Cer+LPS group(P<0.05).2.The overexpression of miR-551b-5p could cause changes in the cytoskeleton and down-regulation of AQP5,Occuldin and Claudin1,thereby increasing the permeability of vascular endothelial cells and exacerbating the capillary leakage and disease of SAP1.After the establishment of the acute inflammatory cell model,qPCR and WB results showed that compared with the NC group,the expression of miR-551b-5p in the acute inflammatory cell model was significantly increased(P<0.05);The mRNA and protein expressions of AQP5,Occuldin,JAM3 and Claudin1 were significantly down-regulated(P<0.05).These results suggest that acute inflammation can lead to the increase of miR-551b-5p expression and the decrease of tight junction-related molecules(AQP5,Occuldin,JAM3 and Claudin1)expression.2.Transient transfection was used to up-regulate and down-regulate miR-551b-5p in endothelial cells.qPCR results showed that the overexpression of miR-551b-5p in the cells was successful(P<0.05),but the down-regulation of miR-551b-5p in the cells was a failure(P>0.05).3.The Lucifer Yellow(LY)permeability test showed that compared with the blank group and control group,the LY permeability of miR-551b-5p mimic group was significantly increased(P<0.05).4.Phalloidin/FITC staining showed that the cytoskeleton of endothelial cells in miR-551b-5p mimic group was relatively loose,with obvious vacuole formation.5.qPCR,WB,and immunofluorescence showed that compared with the control group,the mRNA and protein expression of AQP5,Occuldin,JAM3,and Claudin1 in miR-551b-5p mimic group were significantly decreased(P<0.05).6.In the mouse pre-experiment,the qPCR shows:1)intraperitoneal injection(IP)of AAV8 was more effective than intravenous injection(IV);2)overexpression of miR-551b-5p in mouse pancreas was successful,while down-regulation of miR-551b-5p in mouse pancreas failed.7.ELISA was used to detect serum indexes of mice in each group,and the results showed that compared with the NC group,the expression of serum amylase,lipase,and inflammatory factors(TNF-α,IL-1β and IL-6)in the other groups from high to low was AAV8 miR-551b-5p+Cer+Lps group,AAV8 NC+Cer+Lps group,and AAV8-Mir-551b-5p group.The results suggest that miR-551b-5p can aggravate the condition of SAP mice.8.The capillary leakage and the condition of mice in each group were detected.The results showed that compared to the NC group,the wet/dry weight ratio of lung and pancreas,the concentration of Evan’s Blue in tissue,the severity of pathological changes and the distribution of MPO in other groups were from severe to mild:AAV8 miR-551b-5p+Cer+Lps group,AAV8 NC+Cer+Lps group,AAV8-miR-551b-5p group.It shows that miR-551b-5p can aggravate the condition and capillary leakage of SAP mice.9.qPCR and WB results:Compared with the control group,The mRNA and protein expressions of AQP5,Occuldin,JAM3 and Claudin1 genes in the pancreas tissue of mice in the AAV8-miR-551b-5p group,AAV8 NC+Cer+Lps group,and AAV8-miR-551b-5p+Cer+Lps group were both significantly decreased.Among them,the expression of AQP5,Occuldin,JAM3 and Claudin1 genes in the AAV8-miR-551b-5p+Cer+Lps group decreased most significantly.These results suggest that miR-551b-5p could aggravate the down-regulation of tight junction-related molecules in the SAP mouse model.3.miR-551b-5p could inhibit PI3K/AKT signaling pathway by targeting ERBB3,thereby changing the intercellular junction,leading to increased capillary endothelial permeability and aggravating capillary leakage and disease in SAP.1.A total of 261 differentially expressed genes were obtained by RNA-seq,including 130 up-regulated genes and 131 down-regulated genes.2.Combined with bioinformatics analysis,miR-551b-5p could affect the PI3K/AKT signaling pathway by targeting ERBB3.3.GO and KEGG enrichment analysis of all 261 differentially expressed genes showed that they were mainly enriched in inflammatory factors and inflammation-related signaling pathways,such as IL-17 signaling pathway,MAPK signaling pathway,PI3K/AKT signaling pathway,NF-κB signaling pathway,etc.4.In HUVECs and mouse pancreatic tissue,overexpression of miR-551b-5p could decrease the expression of ERBB3 mRNA.Moreover,dual luciferase reporter assay suggested that miR-551b-5p acted on the 3’UTR of ERBB3.These results suggest that ERBB3 is a target gene of miR-551b-5p.5.In the HUVEC cell line and mouse pancreatic tissue,compared with the NC group,pPI3K and pAKT protein in the miR-551b-5p overexpression group were decreased.These results suggested that overexpression of miR-551b-5p could inhibit the PI3K/AKT signaling pathway.6.In HUVEC cell line,addition of 740Y-P(PI3K activator)could rescue the inhibition of PI3K/AKT signaling pathway and down-regulation of AQP5,JAM3,Occludin,Claudin1 caused by miR-551b-5p overexpression.Conclusions:1.miR-551b-5p is positively correlated with capillary leakage and disease severity in AP mice2.The overexpression of miR-55 1b-5p could down-regulation of AQP5,Occuldin and Claudin1,thereby increasing the permeability of vascular endothelial cells and exacerbating the capillary leakage and disease of SAP.3.miR-551b-5p can inhibit PI3K/AKT signaling pathway by targeting ERBB3,thereby changing the intercellular junction and AQP5,leading to increased capillary endothelial permeability and aggravating capillary leakage and disease in SAP.